An enteric virus can replace the beneficial function of commensal bacteria

Despite significant limitations in the ability to detect and annotate eukaryotic viruses present in the gastrointestinal tract, recent deep sequencing efforts reveal the existence of a complex enteric virome^{3, 4}. Members of this viral component of the intestinal microbiota could include pathogens such as noroviruses that continue to persist after disease is resolved⁵, Anelloviridae and Circoviridae family members that are ubiquitously detected in healthy individuals^6-8, and uncharacterized viruses that display little sequence identity with known viruses^9-11. Also, examination of the enteric virome of rhesus monkeys infected with simian immunodeficiency virus (SIV) suggest that many viruses are present at low levels and kept in check by the immune system¹². Evidence that such viruses in the intestine can contribute to physiology beyond acute diarrheal disease is provided by studies examining MNV, a positive-strand RNA virus of the Caliciviridae family that is endemic in mouse facilities¹³. MNV displays tropism for myeloid cells and can establish persistent infection without causing obvious disease in immunocompetent mice^{13, 14}. We recently demonstrated that persistent infection by the MNV strain CR6 (MNV.CR6) induces intestinal pathologies in mice deficient in the inflammatory bowel disease (IBD) gene Atg16L1¹⁵. This observation that MNV induces inflammatory pathologies in a genetically susceptible host resembles similar observations made with commensal bacteria. However, the bacterial component of the microbiota also provides significant benefit to the host by generating metabolites, promoting the development of the mucosal immune system, and preventing colonization by pathogenic microorganisms¹. It is unknown whether eukaryotic viruses in the intestine interact with the host in an analogous symbiotic manner.

Much of our knowledge on the role of commensal bacteria in mucosal immunity comes from characterization of germfree (GF) mice, which display aberrant intestinal morphology and deficiencies in the lymphocyte compartment due to the absence of bacteria¹⁶. Thus, we utilized GF mice as a reductionist model to determine if MNV infection can provide developmental cues that have been mainly attributed to bacteria. GF mice were mono-associated with MNV.CR6 (GF+MNV) in a gnotobiotic isolator by infecting breeding pairs with 3×10⁶ plaque forming units (PFU) and allowing the virus to be naturally propagated to offspring in subsequent generations, analogous to newborns that inherit commensal bacteria from parents (Extended data Fig. 1a). Despite persistent presence of the virus, there were no signs of overt inflammation in the intestine (Extended data Fig. 1b-d).

While GF mice had thin villi containing few CD3⁺ T cells and narrow crypts, the general appearance of the small intestine (SI) in GF+MNV mice resembled that of conventional mice (Fig. 1a-f). The presence of virus in GF mice also partially restored the number of granules and lysozyme expression in Paneth cells (Extended data Fig. 2a-d). These changes in appearance due to MNV.CR6 were associated with a significant increase in the overall cellularity of the lamina propria (LP) and mesenteric lymph nodes (MLNs) (Fig. 1g, h). Consistent with the ability of certain bacterial species to promote lymphocyte differentiation¹, GF mice had reductions in the numbers of CD4⁺ T cells in the LP and MLNs, CD8⁺ T cells in MLNs, interferon (IFN)-γ expression by these cells, and mucosal and serum antibody production (Extended data Fig. 2e-p). In contrast, GF+MNV mice displayed increases in these factors, in some cases to an extent similar to or in excess of conventional mice (Extended data Fig. 2e-p). These effects of the virus were not dependent on neonatal infection since reversal of abnormalities was observed when adult GF mice were infected with MNV.CR6 for 10 days (Extended data Fig. 3). Also, the effects of MNV.CR6 cannot be explained by uncontrolled viral replication because GF mice harbor less virus compared to conventional mice upon infection (Extended data Fig. 1b), as seen with other intestinal viruses^{17, 18}. Importantly, these changes in response to MNV.CR6 are specific to conditions in which bacteria are absent. MNV.CR6 had little or no effect on the appearance of the intestine and lymphocytes in conventional mice (Extended data Fig. 4). Therefore, there is significant overlap in responses to MNV and commensal bacteria.

The host response evoked by MNV could depend on anatomical location or the strain examined. Abnormalities in the morphology of the colon of GF mice were not as apparent as in the small intestine, but we detected a significant increase in NKT cells¹⁹. While MNV mono-association had little effect on tissue morphology or the number of NKT cells, the presence of the virus increased the frequencies of both CD4⁺ and CD8⁺ T cells in the colon (Extended data figure 5). To determine whether the effect of MNV is specific to the CR6 strain, we infected GF mice with MNV.CW3, a strain that is considered more relevant to human noroviruses because it displays increased virulence compared to MNV.CR6 and establishes an acute infection²⁰. Unlike conventional mice that clear the virus between days 5-7 post-infection²⁰, we found that a low amount of MNV.CW3 can be detected in stool of GF mice on day 10 post-infection (Extended data Fig. 1b), and was able to induce similar effects on intestinal morphology and the lymphocyte compartment as MNV.CR6 (Extended data Fig. 6b-h). Next, we isolated virus directly from a naturally infected mouse housed in the vivarium (MNV.SKI, described in Extended methods section). Alignment of the capsid sequence determined that MNV.SKI is a previously uncharacterized strain (Extended data Fig. 6a). MNV.SKI was also able to reverse abnormalities in GF mice (Extended data Fig. 6b-h). Although all three MNV strains induce qualitatively similar alterations to intestinal morphology and lymphocytes, the effects are quantitatively distinct, especially when comparing T cell subsets. Nevertheless, the general ability of MNV to evoke a response in GF mice appears to be strain-independent.

Innate lymphoid cells (ILCs) are emerging as important participants in mucosal immunity, but the role of the microbiota in maintaining this compartment is less understood. Unlike the skin and lungs, the intestine has been shown to contain reduced numbers of Gata3⁺ ILC2s relative to Rorgt⁺ ILC3s²¹. We found that GF mice had an increase in the proportion of ILC2s in the SI compared to conventional mice (Fig. 1i-m). This expansion of ILC2s due to the absence of bacteria was reversed in GF+MNV mice (Fig. 1i-m). Consistent with this finding, MNV.CR6 infection increased the proportion of ILCs expressing IL-22 relative to IL-13, effector cytokines expressed by ILC3s and ILC2s respectively (Fig. 1i, m). The proportion of the T-bet⁺ ILC1 subset was similar across conditions (Extended data Fig 2q). Thus, we find that commensal bacteria prevent expansion of intestinal ILC2s, and MNV.CR6 infection mimics this function of commensal bacteria. When taken together, these results indicate that the presence of a single virus can reverse many of the abnormalities that arise in the complete absence of the commensal bacterial community (Fig. 1n).

GF mice lack bacteria from birth and represent an extreme condition. We found that adult mice treated with a cocktail of antibiotics (ABX) for 2 weeks display several of the abnormalities described above in GF mice including aberrant intestinal morphology and a reduction in the number of total CD4⁺ Tcells as well as IFN-γ-expressing CD4⁺ and CD8⁺ T cells (Fig. 2a-j and Extended data Fig. 7a-d). Thus, we examined the ability of MNV infection to reverse these abnormalities induced by ABX treatment as another assay to demonstrate functional redundancies between this virus and commensal bacteria (Fig. 2a, Extended data Fig. 1b). By day 10 post-infection, MNV.CR6 infection increased villi width, Paneth cell granules, intestinal T cells, and IFN-γ expression by CD4⁺ T cells (Fig. 2c-m). For comparison, mice were treated with ABX for 2 weeks and then inoculated with 1×10⁷ colony forming units (CFU) of representative commensals Bacteroides thetaiotamicron (B. theta), Lactobacillus johnsonii, or left alone after ABX removal (Extended data figure 7e-h). B. theta and L. johnsonii restored the morphology of the intestine to a similar degree as MNV.CR6, but had varying effects on the T cell compartment. B. theta increased T cell numbers when measured by CD3-staining of small intestinal sections while L. johnsonii induced an IFN-γ response in CD4⁺ and CD8⁺ T cells without affecting their overall numbers (Extended data Fig. 7i-p). These results are consistent with the concept that individual members of the microbiota evoke overlapping responses from the host, but are not necessarily identical.

To gain better resolution into the response evoked by MNV and commensal bacteria, we performed RNA deep sequencing analyses (RNA-seq) on whole intestinal tissue harvested from untreated GF mice, GF mice 10 days after inoculation with MNV.CR6, and GF mice reconstituted with the bacterial flora from conventional mice (Fig. 3a, Extended data table 1). Pathway analysis of the transcripts induced by commensal bacteria indicated alterations in the metabolic state, immunity, and blood vessel morphogenesis. MNV.CR6 induced a more limited gene expression pattern associated with lymphoid cell development and immune responses, many of which are related to the antiviral type I interferon (IFN-I) response (Extended data table 1a). Consistent with the shared ability of MNV and bacteria to reverse abnormalities in GF mice, the overlapping gene set contained genes associated with development and function of hematopoietic lineage cells (Fig. 3a, Extended data table 1c). The IFN-I signature induced by MNV.CR6 prompted us to examine whether the effect of viral infection was dependent on IFN-I signaling. The intestine of untreated IFNα receptor knockout mice (IFNAR^−/−) displayed a normal appearance, while ABX-treatment of these mice led to similar abnormalities in intestinal morphology and T cell numbers as described above (Fig. 3b-h). However, MNV.CR6 infection did not have a detectable effect on ABX-treated IFNAR^−/− mice despite productive replication (Fig. 3i, Extended data fig. 1b), indicating that virus-mediated reversal of these abnormalities is dependent on IFN-I signaling. Inducing IFN-I gene expression through poly(I:C) injection was sufficient to increase villus width in GF but did not have a noticeable effect on intestinal T cells (Extended data figure 8). Thus, IFN-I signaling likely functions in conjunction with other pathways to confer the full effect of MNV infection. Indeed, transcriptional analyses supports a large overlap between the genes induced by MNV.CR6 and bacterial colonization of GF mice that are not associated with IFN-I (Extended data table 1c).

Depletion of commensal bacteria is considered a major health hazard of indiscriminate use of antibiotics. Because MNV can reverse abnormalities observed upon bacterial depletion, we tested the possibility that viral infection can provide protection against intestinal damage. We found that ABX-treatment increased sensitivity to chemical injury of the intestine by dextran sodium sulfate (DSS) (Fig. 4a). Remarkably, all three MNV strains examined were able to improve survival in ABX-treated mice receiving DSS (Fig. 4a). MNV.CR6 in particular conferred robust protection and prevented shortening of the colon (Fig. 4a, b). MNV.CR6 was unable to improve survival in ABX-treated IFNAR^−/− mice indicating that IFN-I signaling was necessary (Figure 4c). Next, we used inflammation induced by Citrobacter rodentium to test the protective effect of MNV because ABX-pretreatment was shown to exacerbate disease induced by this model enteric pathogen without altering bacterial burden²². In ABX-pretreated mice, MNV.CR6 did not alter the amount of C. rodentium, which was the dominant bacterium in the gut (Fig. 4f, Extended data fig. 9a-b). However, MNV.CR6 ameliorated weight loss, diarrhea, and histopathology (Fig. 4d-k). C. rodentium induces expression of virulence factors in response to host immunity and competition with other bacteria²³. Interestingly, MNV infection of ABX-treated mice restored expression of bacterial virulence factors to levels detected in conventional mice (Extended data fig. 9c-d). This effect of the virus on bacterial gene expression and the protection from inflammation are most likely due to the ability of MNV.CR6 to enhance the immune response to C. rodentium, which includes increased levels of antibodies as well as antimicrobial and regulatory cytokines (Extended data fig. 9e-i). Thus, MNV enhances protection against both infectious and non-infectious secondary challenges to the gastrointestinal tract in ABX-treated animals.

RNA viruses are routinely detected in asymptomatic infants and children as well as individuals recovering from acute gastroenteritis^{5, 24, 25}. Thus, in addition to periods following ABX-administration, the presence of known or unidentified viruses may be beneficial during developmental or recovery stages when the immune system and the intestinal microbiota are vulnerable. However, like bacteria, whether a given intestinal virus is a mutualist is likely to depend on host factors and the specific viral strain in question. For instance, production of IFN-I is a conserved response to viral infections, but the role of this cytokine in the gut is highly contextual and dependent on other soluble factors²⁶. For this reason, it may not be surprising that MNV can protect ABX-treated mice from intestinal injury, but exacerbates disease in mice that are genetically susceptibility to IBD^{15, 27}. We speculate that viruses that contribute to the pathogenesis of complex inflammatory disorders could be commensal in nature rather than exclusively pathogenic. Taken together, we demonstrate that an enteric animal virus can function in a manner analogous to commensal bacteria, and propose that symbiotic viruses exist in the mammalian gastrointestinal tract. We suggest that the incredible advances in viral discovery pipelines²⁸ be leveraged to discover commensal or mutualistic viruses that constitute the virome in addition to traditional pathogens.